Istituto Superiore di Sanità, Roma

Reparto di Neuroscienze ComportamentaliReparto di Neurotossicologia e NeuroendocrinologiaDipartimento di Biologia Cellulare e Neuroscienze

Ricerca di base su modellisperimentali di disturbi dello spettro autistico in ISS (1) : Analisi neurocomportamentali

Laura Ricceri Gemma Calamandrei

Ringraziamenti:

• Maria Luisa Scattoni, Ph.D.

• Jacqueline Crawley, Lab. Behavioral Neurosciences, NIMH• ISS-NIH Collaborative Program

Eterogeneità e multifattorialità nelle sindromi dello spettro autistico

In verde: eterogeneità clinicaIn azzurro: eterogeneità genetica e ambientaleAl centro: disfunzioni del neurosviluppo responsabili di un’alterata funzionalità sinaptica

Causes of Autism Spectrum Disorders: strongest evidence is genetic

a) 4:1 frequency ratio boys:girls

b) >60% concordance for monozygotic twins

c) Linkage analyses indicate multiple genes underlying this complex disease, including candidate genes at loci on chromosomes 6, 7, 13, 15, 17

d) Proposed candidate genes include GABA receptor subunit GABRB3 Neuroligin 3 and 4, FOXP2, WNT2, HOXA1, engrailed-2, Fragile X, serotonin transporter polymorphisms, Reelin and other developmental genes and transcription factors

Loci implicated in ASD etiology (from Abrahams and Geschwind, 2008)

(from Persico and Bourgeron, 2006)

•From the dominance of psychodynamic theories ofautism as recently as 1970 to the awareness ofupwards of 20 bona fide risk genes today, it must beconcluded that substantial progress has been made. That the identification of virtually every ASD-relatedgene and syndrome occurred within the last 5–10 years is particularly telling.

•Linkage studies have not found ‘the autism gene’but have unequivocally demonstrated that more sophisticated solutions will be required to explain thisgroup of disorders. The availability of new technologyis likely to provide more satisfying answers.

•Despite the immense amount of work implied in thisagenda, the landscape for the future study of the ASDs is coming into view and has never looked aspromising.

(from Abrahams and Geschwind, 2008)

modelli animali: a cosa servono

• valutazione delle relazioni genotipo-fenotipo di singoli fattori genetici

• studio dei meccanismi

• analisi delle interazioni tra fattori genetici e singoli fattori ambientali

• determinazione di periodi critici di suscettibilità

• valutazione preclinica di possibili strategie terapeutiche

modelli animali:

Manipolazione genetica o mutazione

spontanea in un singolo gene

[Somiglianza neimeccanismipatogenetici

(construct-validity)]

+ NLG3 etc

Ceppi di topi (inbred mouse

strain) che mostrano un particolare

fenotipo comportamentale[Somiglianza dei

sintomi (face-validity)]

BTBR T+tf/J mice

modelli animali:

Autism is a neurodevelopmental disorder that is behaviorally defined by three core symptoms:

1. Deficits in reciprocal social interaction

2. Stereotypies, repetitive, ritualistic behaviors, narrow restricted interests

3. Qualitative impairments in social communication

Animal Models of AutismCore Symptom #1

SociabilitySociability

Most mice will approach a new conspecific, spend a lot of time together, and display

high levels of investigative sniffing

Automated Social Approach Apparatus

subjectsniffs

stranger 1

retractable partitionsbetween chambers

centralstart

chamber

Empty wire cage

control

photobeam motiondetectors across

entryways

Designed and fabricated by Jacqueline Crawley, NIMH, Sheryl Moy, UNC,George Dold and coworkers,

NIMH/NINDS Research Services Branch, Bethesda, MDc

Yang M, Frontiers in Behavioral Neuroscience 2007

Social Deficit

BTBR B6

Tim

e in

Sid

e C

ham

ber(

sec)

0

100

200

300

400

500 Novel objectCenterStranger Mouse

**

A

BTBR B6

Tim

e Sn

iffin

g (s

ec)

0

100

200

300

400Novel ObjectStranger Mouse

**

B

Juvenile Play Behaviors

Noldus Observer Phenotyperwith digital videocamera, keypad event recorder,

and frame-by-frame data analysis software

Pairs of 20 day old juvenile male mice of the same strain but from different home cagesare placed in the arena for a30 minute play session. Eventsscored include social approach, social grooming, following,crawling over/under,etc.

Measures of non-socialgrooming, exploration andactivity are simultaneouslyscored.

One mouse grooms the otherSocial Grooming

McFarlane HG, G2B 2007

Nose-to-Nose SniffingOne mouse sniffs the snout

region of the other

Freq

uenc

y

Inactive AloneBoth mice sit at rest, separate

from each other,neitherexploring nor self-grooming

Core Symptom #2Stereotyped Repetitive BehaviorsStereotyped Repetitive Behaviors

Videoscoring of spontaneous stereotyped grooming

Core Symptom #3Impairment in Social CommunicationImpairment in Social Communicationdelayed onset and lower frequency of babbling, unusual gestures, diminished responsiveness,

and desynchronization of vocal patterns in humans

Mice communicate primarily with olfactory pheromones and ultrasonic vocalizations

Mouse ultrasonic vocalizations

Pups produce whistle-like sounds (frequency range between 40 and 90 kHz) that elicit maternal care.

These calls are structurally similar to cries and nonverbal sounds of human infants.

Ultrasonic Vocalizations

Scattoni et al, PlosOne 2008

BTBR pup

B6 pup

File name C0000027

File name: B0000029

Spectrogram differences

Scattoni et al, PlosOne 2008

Chevron

10203040506070

kHzHarmonic

Short Two-syllable

Upward

Flat Composite Frequency step

Downward Complex

Ten categories of mouse pup calls

Call distributions: 4 inbred strains

Chevron Complex

Composite

Downward

Flat

Frequency stepsHarmonics

Short Two-syllables

Upward

B6

16%12%

5%

14%

7%31%

3%

8%

1%3%

BTBR

1%

8%

18%

3%5%23%

29%

1% 12%0%

129/SvJ

2%17%

9%

3%5%

45%

1%

3%

14%1%

FVB/NJ

6%17%

11%

4%

6%

43%

1%

3%7%

2%

Scattoni et al, PlosOne 2008

• Incorporate robust behavioral phenotypes for all 3 symptoms  of autism

– Reduced sociability (social approach, juvenile play, social interactions)

– Repetitive behaviors (greater self‐grooming) – Communication deficits (unusual repertoire in ultrasonic 

vocalizations)

encourage its use as a research tool forneurophysiological investigations (e.g.

modulations of the behavioral phenotype by hormonal milieu)

extension of such behavioral phenotypingmethods (qualitative USV analysis) to other

ASD mouse models

BTBR T+tf/J mice

Mouse pup

ThermalOlfactoryTactileSocial

MOTHER& littermates

ULTRASONIC VOCALIZATIONS (USVs) EMITTED BY INFANT RODENTS

ISOLATION

Ultrasonic emission

1024 2048 ms

36

48

60

Khz

- ultrasonic vocalizations of infant mice are whistle-like sounds emitted mainly in the frequency range between 40 and 90 kHz

- these calls are associated with social isolation, they have been described as distress vocalizations or isolation calls